Protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step immunoprecipitation approach.

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses. For complete details on the use and execution of this protocol, please refer to Li et al.1.

ATG5 nonautophagically regulates inflammation and differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs), with abilities of infinite proliferation (self-renewal) and to differentiate into distinct cell types (pluripotency), show attenuated inflammatory response against cytokines or pathogens, which is recognized as a unique characteristic of ESCs compared with somatic cells. However, the underlying molecular mechanisms remain unclear, and whether the attenuated inflammatory state is involved in ESC differentiation is completely unknown. Our recent study demonstrated that macroautophagy/autophagy-related protein ATG5 inhibits the inflammatory response of mouse ESCs (MmESCs) by promoting the degradation of BTRC/ß-TrCP1 and further the downregulation of NFKB/NF-κB signaling. In addition, maintenance of an attenuated inflammation status in MmESCs is required for their differentiation. In conclusion, ATG5 is a key regulator for the regulation of inflammatory response and differentiation of MmESCs.